Natural allelic variation confers diversity in the regulation of flag leaf traits in wheat

Flag leaf (FL) dimension has been reported as a key ecophysiological aspect for boosting grain yield in wheat. A worldwide winter wheat panel consisting of 261 accessions was tested to examine the phenotypical variation and identify quantitative trait nucleotides (QTNs) with candidate genes influencing FL morphology. To this end, four FL traits were evaluated during the early milk stage under two growing seasons at the Leibniz Institute of Plant Genetics and Crop Plant Research. The results showed that all leaf traits (Flag leaf length, width, area, and length/width ratio) were significantly influenced by the environments, genotypes, and environments × genotypes interactions. Then, a genome-wide association analysis was performed using 17,093 SNPs that showed 10 novel QTNs that potentially play a role in modulating FL morphology in at least two environments. Further analysis revealed 8 high-confidence candidate genes likely involved in these traits and showing high expression values from flag leaf expansion until its senescence and also during grain development. An important QTN (wsnp_RFL_Contig2177_1500201) was associated with FL width and located inside TraesCS3B02G047300 at chromosome 3B. This gene encodes a major facilitator, sugar transporter-like, and showed the highest expression values among the candidate genes reported, suggesting their positive role in controlling flag leaf and potentially being involved in photosynthetic assimilation. Our study suggests that the detection of novel marker-trait associations and the subsequent elucidation of the genetic mechanism influencing FL morphology would be of interest for improving plant architecture, light capture, and photosynthetic efficiency during grain development.


Plant material and field trials
A worldwide winter wheat panel consisting of 261 accessions was tested to examine the natural phenotypical variation of flag leaf-related morphology.Seeds from the whole panel were provided by the German Federal ex-situ Genebank located at the Leibniz Institute of Plant Genetics and Crop Plant Research (Gatersleben, Germany).Schierenbeck et al. 33,34 and Supplementary Table 1 reported more information on the winter panel.The authors comply with the IUCN policy statement on research involving species at risk of extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.All methods were carried out in accordance with institutional, national, and international relevant guidelines and regulations.
Field trials were performed at the Leibniz Institute of Plant Genetics and Crop Plant Research during 2016-2017 and 2018-2019 following a randomized complete blocks design with three replications.Each accession was sown in 2.4 m 2 plots (2 m long by 1.2 m wide) and contained six rows.Four flag leaf traits were measured during the early milk stage (Z73) 35 , assessing twenty random of plants per plot.FLL (flag leaf length; distance from the base to the tip of the leaf) and flag leaf width (FLW; distance from the widest part of the leaf) were measured as suggested by Liu et al. 36 , while flag leaf area (FLA = FLL × FLW × 0.75) and length/width ratio (FLWR = FLL/FLW) were calculated based on Yang et al. 19 .

Genotyping and population structure
A chip of the 90 K iSELECT 42 was used to genotype the population.After the quality check, 17,093 SNP markers were mapped according to their physical position based on IWGSC RefSeq v2.1 (http:// www.wheat genome.org/) and then used to calculate the population structure, linkage disequilibrium (LD), and GWAS scan.The PCA analysis showed that the panel clustered into three groups strongly according to their different origins: 66 genotypes (25.2%) from Central-Northern Europe mainly Germany, France, Poland, Sweden, Finland, and Great Britain; 146 genotypes (55.6%) from Eastern Europe-Western Asia like Russia, Bulgaria, Kazakhstan, Ukraine; 42 accessions (16%) from North-America mainly from USA and Canada.The remaining genotypes (3.2%) come from diverse countries worldwide (Australia, Chile, China, Japan, and India).Regarding the marker coverage, the B genome showed the highest density with 8809 SNPs (51.5%), followed by the A genome (38.6% of all markers, 6595 SNPs) and the D genome with 9.9% (1689 SNPs).The homoeologous chromosome of group 1 had the highest number of SNPs (17.96%), while the chromosomes of group 4 had only ca.7%.Chromosome 5B had the highest number of SNPs with 1784 markers, while chromosome 4D had only 46 SNPs.More details of the population were published in Refs. 33,34.

Genome-wide association study and identifying putative candidate genes
In the current analyses, we used the FARM-CPU model through GAPIT 3 in the R environment 43 .This model was selected due to improved statistical power, efficient computing time, and prevention of model overfitting compared to other models 44 .The GWAS analyses were calculated for each environment separately and BLUE values over the environments.If the − log 10 (p-value) of SNP passed the threshold of − log 10 (1/number of SNP markers = 5.85e −5 ) = 4.23, the SNP was considered as a significant association QTN and used for further analysis 45,46 .
The significant QTNs that were present in the two environments were further used to identify the highconfidence (HC) putative candidate genes within an LD ± 2 Mbp interval.We used the latest version of the wheat reference genome sequence of Chinese Spring by blasting against IWGSC RefSeq annotation v2.1 (http:// www.wheat genome.org) to identify the accurate physical position of QTNs and candidate genes.Because each block of LD contains a high number of candidate genes, we have selected those that have SNPs within their physical positions.To get more molecular and cellular knowledge about these genes, the WheatMine platform was used to search for the gene ontologies (GO) and InterPro number and description (https:// urgi.versa illes.inra.fr/ Wheat Mine/ begin.do).The underlying genes were further examined for their association with flag leaf morphology traits using previously published literature.
Expression analysis has been done through the RNA-Seq expression data from the Wheat Expression database (https:// bar.utoro nto.ca/ eplant_ wheat/) 47 which includes the expression of genes for flag leaf and grain development.Gene expression is presented as TPM (Transcripts Per Kilobase Million).

Phenotypic variations on flag leaf dimension traits
Flag leaf morphological traits were significantly influenced by the environments, genotypes, and Environment × Genotypes interactions (Table 1).Variations across different environments and summary statistics are shown in Table 2 and Fig. 1.Data analysis revealed extensive natural phenotypic variation with the normally distributed FL traits suggesting the suitability of the studied traits in the used association panel for additional genetic studies.A broad-sense heritability ranging from 0.72 to 0.94 was found which demonstrates that the traits are predominantly genetically controlled.Correlations among flag morphology in the different growing seasons are shown in Fig. 2. Low or non-significant correlations were reported between FLL and FLW.FLA showed a high correlation with FLL and FLW, but a slightly better adjustment with FLW (ca.0.76).For their part, negative correlations were detected for FLWR and FLW, while minor negative correlations (ca.− 0.20) were documented for FLWR and FLA.The diverse origins of the genotypes presented a differential response to FL morphology traits.Cultivars from Central and Northern Europe exhibited higher values for FLW and FLA (1.61 cm and 24.74 cm 2 , respectively) in comparision with North American cultivars (1.31 cm and 19.56 cm 2 , respectively).Genotypes from Eastern Europe-Western Asia showed intermediate values (Fig. 3).

Candidate genes underlying flag leaf morphology traits in winter wheat
After detecting stable QTNs a further analysis exhibited eight novel high-confidence candidate genes influencing flag leaf morphological traits on chromosomes 2A (2), 3A, 3B (2), 5A and 6B (2) (Table 3).Based on GWAS and LD outputs, we mined the most reliable candidate genes affecting FL dimensions (Supplementary Figs.S1 to S6).
For example, on chromosome 3A a strong and significant QTN (RAC875_c52195_324) was detected and located inside exon 2 of the TraesCS3A02G480600 gene at position 711401772-711407423 bp (Supplementary Fig. S1a, Table 3).The candidate gene encodes the BTB/POZ domain (IPR000210) which harbors the phenotypic variation of FLL (Supplementary Fig. S1b).For this QTN, allelic effects showed longer flag leaf length for genotypes carrying the A allele compared to G, demonstrating that those varieties would be used as elites for markerassisted selection (Supplementary Fig. S1c).
Interestingly, our study identified a highly significant SNP, namely wsnp_RFL_Contig2177_1500201 at chromosome 3B (Supplementary Fig. S2a).This SNP located on exon 6 of gene TraesCS3B02G047300 at position 23957233-23962495 bp, encodes a Major facilitator, sugar transporter-like (IPR005828), which harbors variations on FLW (Supplementary Fig. S2b).Moreover, the allelic effect for this QTN showed longer FLW for genotypes carrying the G allele, demonstrating that those genotypes would accumulate more sugars via the photosynthesis process that in turn may lead to increases in kernel weight (Supplementary Fig. S2c).
Another significant SNP, namely RAC875_rep_c112818_307 was located on chromosome 5A (Supplementary Fig. S3a).This SNP located inside exon 4 of the TraesCS5A02G428800 gene at position 613477485-613482016 bp, encodes Synaptobrevin that also harbors variations on FLW (Supplementary Fig. S3b).Further, accessions carrying the G allele showed a positive effect on flag leaf width compared to accessions carrying the A allele (Supplementary Fig. S3c).
The QTN IAAV873 was located on chromosome 6B and was associated with FLW.This QTN was located within exon 1 of TraesCS6B02G251400 at position 450644047-450644247 bp which encodes the MATH/TRAF domain (Supplementary Fig. S4a,b).The TRAF domain has a novel positive contributor to plant immunity that The expression analysis of the eight high-confidence candidate genes showed a wide range of gene expression (Fig. 5).High expression values in flag leaves and interestingly also in grain development in different developmental stages were reported for TraesCS3B02G047300, TraesCS2A02G464100, TraesCS5A02G428800, and TraesCS3A02G248900 while intermediate values were detected for TraesCS6B02G353200, TraesCS2A02G029800, TraesCS6B02G251400, and TraesCS3A02G480600.

Phenotypic variation
Exploring genotypic variations and uncovering the genetic basis of flag leaf architecture traits have been considered key traits for increasing photosynthesis efficiency and grain yield potential 17 .The current study revealed extensive phenotypic variation in flag leaf traits across the 261 winter wheat accessions, with high heritability reported for all traits.Furthermore, a high correlation among environments was observed, suggesting that these traits are predominantly genetically controlled.Coinciding with Ref. 27 , increasing FLW is an effective approach for FLA enhancement compared to FLL.Previous reports using association panels and biparental populations also documented high heritabilities and positive correlations among flag leaf morphological traits and grain yield parameters 21 suggesting FL size optimization as an appropriate breeding approach for increasing wheat yield potential 20 .
The origins of the accessions used in the study were documented to exhibit a diverse range of flag leaf morphology traits, thereby demonstrating the diversity of such traits.Cultivars from Central and Northern Europe showed higher values for FLW and FLA compared to North American and Eastern Europe-Western Asia accessions.Extensive phenotypic variation among these traits has been widely reported mainly in DH and RIL populations 16,22 and diverse panels 17,30 , however, an association on the genotype´s origin as described here has not been previously documented.Future studies should assess if differences in FL dimensions are related to an indirect selection effect concerning the photosynthetic active radiation available at different latitudes.

Novel QTNs and Candidate genes linked with flag leaf morphology
We reported 10 novel and stable QTNs (LOD ≥ 4.23) in at least two environments related to flag leaf dimensions over seven chromosomes of the wheat genome (1A, 2A, 3A, 3B, 4B, 5A, 5B, and 6B).As previously reported, flag leaf morphological traits are quantitative traits controlled by multiple genes 6,65 .Recently some authors have compiled previous reports related to QTL involved in flag morphological traits on different wheat chromosomes 22,65 .Even though previous efforts mainly using RILs and DH population have documented markers linked with flag leaf morphology on 1A 22 , 1B 19,36 , 2A, 19 , 2B 19 , 3A 36 , 3B 22 , 4A 22 , 4B 22 , 5A 19,36 , 5B 19 , 6B 36 , 7A 22 , 7B and 7D 36 , those www.nature.com/scientificreports/QTNs documented in this work have not been previously reported.These findings would indicate the potential of these novel QTNs for improving flag leaf architecture and therefore, boosting light capture and photosynthetic efficiency during grain filling.
In the same sense, a further bioinformatics analysis revealed eight novel candidate genes influencing flag leaf morphology on chromosomes 2A (2), 3A, 3B (2), 5A, and 6B (2).Although these candidate genes have been documented for diverse traits in wheat (such as disease resistance, grain quality traits, grain yield, plant growth Table 3. Distribution of significant QTN (quantitative trait nucleotides) in at least two environments and candidate genes associated with flag leaf morphology traits.Significant threshold − log 10 (5.85e −5 ) = 4.23.Chr (Chromosome); cM (Centimorgan); FLL (flag leaf length); FLW (flag leaf width); FLA (flag leaf area); FLWR (flag leaf length/width ratio); LOD: logarithm of the odds; NA (no information available).www.nature.com/scientificreports/and development) none of them have been reported to be associated with traits related to flag leaf morphology in wheat, which highlights the importance of our work (Table 3).A significant QTN (RAC875_c52195_324) was detected on chromosome 3A and located inside the TraesC-S3A02G480600 gene at position 711401772-711407423 bp.This gene encodes the BTB/POZ domain (IPR000210) which controls the phenotypic variation of FL length.Our results revealed that the allelic variation for marker detected that the accessions carrying A allele (229 genotypes) presented longer flag leaves compared to genotypes carrying the G allele (32 genotypes) (Supplementary Fig. S1).A recent study by 22 detected 2262 putative genes related to flag leaf size within the MQTL regions which mainly encode the F-box-like domain proteins, protein kinases, and BTB/POZ domain-containing proteins, suggesting their crucial roles in regulating leaf growth and development in Arabidopsis.Shariatipour et al. 66 also reported eight wheat homologs for rice genes located on the MQTL-4B.5, MQTL-5B.3,MQTL-7A.1,MQTL-7B.1, and MQTL-7D.2, respectively, demonstrating the implication of these homolog genes in various biological processes associated with leaf size and chlorophyll content in rice and suggesting their involvement in the regulation of leaf size in wheat.Moreover, the effect of RAC875_c52195_324 on wheat grain quality traits has been also reported 53 , the response that would be associated with the expression levels here reported for the candidate genes during grain filling (Table 3; Fig. 5b).
Interestingly, our study identified a highly significant SNP, namely wsnp_RFL_Contig2177_1500201 on chromosome 3B.This QTN has also been reported to play a role in leaf rust resistance (hcmQTL3B.2) 56 and grain yield-related traits (MQTL3B.1) 57(Table 3).This SNP is annotated as the candidate gene TraesCS3B02G047300 and encodes a major facilitator, sugar transporter-like (IPR005828) that harbors the variation of the FLW.The allelic variation for this marker showed that cultivars with the G allele (33) showed wider flag leaves compared to those carrying the A allele (228 genotypes) (Supplementary Fig. S2).The sugar transporter proteins (STPs) play www.nature.com/scientificreports/important roles in plant growth development, signal transmission, and cellular ion homeostasis under certain biotic and abiotic stress tolerance 67 .To date, it has been established that sugars are produced by photosynthesis and distributed mainly as sucrose through the phloem to other parts of the plant.Some of the sucrose is unloaded directly into the sink organs via the symplast whereas other sucrose is carried over long distances to the sink organs.Sucrose invertase breaks down sucrose into glucose and fructose, which produces apoplastic sugar, which is then absorbed through transmembrane absorption and transported to the sink cells by sugar transporters (STP) 68 .Since the distribution of sugars between assimilate-exporting source tissues and sugar-consuming sink tissues is crucial for plant growth and development, sugar transport in plants is considered an important research topic with economic significance for a food-secure world.This response could explain the higher expression values among all the candidates genes reported in flag leaf tissues and grain development reported here for this candidate gene (Fig. 5a,b).In Arabidopsis, more than 50 MSTs were identified and the STP subfamily is comprised of 14 monosaccharide/H + symporters 69 .The STP subfamily encodes H + -importing monosaccharide transporters, which can transfer diverse hexoses and/or pentoses but not sucrose 70 .One of the main roles of STPs, which are almost all high-affinity hexose transporters with specialized expression in tissues, is to increase the sink needed for photosynthate redistribution 69 .Huai et al. 71 reported that the ABA-induced sugar transporter TaSTP6 increases sugar supply and promotes fungal infection in wheat.In Arabidopsis, the expression of STP13 is greatly increased when challenged with Botrytis cinerea 72 .In maize, expression of Sucrose Transporter 1 (ZmSUT1) is enhanced in response to the biotrophic and necrotrophic development of the pathogen Colletotrichum graminícola 73 .Taken together, these processes are essential for preserving source/sink characteristics and hormonal signals, which play an important role in the whole plant development, cell growth, especially leaf-related traits, and osmotic homeostasis under certain biotic and abiotic challenges.Another significant SNP, namely RAC875_rep_c112818_307, was located on chromosome 5A and has been previously related to spike fertility 61 , number of spikes 62 and leaf rust resistance 63 .This SNP, annotated as the candidate TraesCS5A02G428800 at position 613477485-613482016 bp, encodes Synaptobrevin which harbors the variation of the flag leaf width.We reported that genotypes carrying G allele (240 genotypes) presented wider flag leaves compared to those with A allele (21 genotypes) (Supplementary Fig. S3).Wang et al. 74 identified TaSYP137, an R-SNARE subfamily gene, and TaVAMP723, from wheat as having long synaptobrevin domains.An evolutionary analysis of the genes TaSYP137 and TaVAMP723 shows that they are closely genetically related to XP_037417660.1 and XP_037439902.1,with high degrees of similarity to homologous proteins in other species.This suggests that the TaSYP137/TaVAMP723 protein may perform similar biological functions in plant development and response to biotic and abiotic stresses, similar to the corresponding proteins in other species.
Ultimately, the QTN IAAV873 is located on chromosome 6B and was associated with flag leaf width and annotated as TraesCS6B02G251400 at position 450644047-450644247 bp which encodes the MATH/TRAF domain.For this marker, genotypes carrying A allele (151 genotypes) presented wider flag leaves compared to those carrying the C allele (110 genotypes) (Supplementary Fig. S4).The tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) domain, also referred to as the meprin and TRAF-C homology (MATH) domain, is a protein-protein interaction structure that can be found in diverse species 75 .Immune receptors play important roles in the perception of pathogens and the initiation of immune responses in both plants and animals.A study by Ao et al. 76 identified mutations in TRAF CANDIDATE 1b (TC1b), a gene encoding a protein with four tumor necrosis factor receptor-associated factor (TRAF) domains that were shown to suppress snc1 phenotypes underlying immune receptors.TC1b also does not physically associate with SNC1, affect SNC1 accumulation, or affect signaling of the downstream helper NLRs represented by ACTIVATED DISEASE RESISTANCE PROTEIN 1-L2 (ADR1-L2), suggesting that TC1b uniquely impacts snc1 autoimmunity.Overall, uncovering the TRAF domain protein TC1b as a novel positive contributor to plant immunity is of high importance for improving plant growth and development via regulating photosynthetic assimilation in flag leaf.
This study provides desirable alleles for FL optimization and is useful for wheat breeding strategies.In this sense, we further described that the allelic variation within the CG associated with flag leaf morphology affects several cellular and metabolic processes, leading to effects on leaf growth and senescence regulation, chlorophyll content, and prolongation of the grain-filling period.Future studies will seek to expand the set of genotypes and to utilise novel multilocus GWAS models with the objective of reducing the computation time and increasing the accuracy of QTN detection 18 .Moreover, the detection of novel QTNs and the subsequent elucidation of the genetic mechanism influencing flag leaf dimensions would be of interest for improving plant architecture, radiation capture, and photosynthetic efficiency during grain filling.

Conclusions
Due to its role as the main contributor of assimilates for grain filling, flag leaf architecture has been reported as a main ecophysiological trait for boosting grain yield potential.High natural variation in leaf traits plays a vital role in improving grain yield and environmental stress adaptation.Using a worldwide winter wheat panel, ten stable and novel QTNs were detected playing a role in controlling flag leaf morphology.Furthermore, new candidate genes were reported to be likely involved in these traits, showing high expression values in flag leaf tissues through the crop cycle and interestingly, also during grain development.The obtention of novel QTNs linked and the subsequent elucidation of the genetic mechanism related to light absorption capture and their close relation with photosynthate assimilation at the grain filling would be of interest for marker-assisted selection in wheat breeding programs.

Figure 2 .
Figure 2. Correlation among flag leaf dimension traits in wheat genotypes.The degree of significance for all correlations across different years was P < 0.001.The color reflects the strength of the correlation.Nonsignificant correlations are expressed using crosses.

Figure 4 .
Figure 4. Manhattan plots showing significant marker trait associations for (a) Flag leaf length, (b) Flag leaf width, (c) Flag leaf area and (d) Flag leaf lenght/width ratio in 261 winter wheat genotypes.For each trait, dashed red lines and highlighted black circles show QTNs surpassing the significant threshold − log 10 (5.85e −5 ) = 4.23 in two environments.Names of significant QTNs are pointed with black arrows.